3D organoid cultivation improves the maturation and functional differentiation of cholangiocytes from human pluripotent stem cells

Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC–derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system–generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.


Supplementary Table
: List of reagents used in cholangiocyte differentiation from hPSCs in 2D and 3D culture system.Cultivate the human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using proprietary "Primary ES Medium" and supplemented with 12 ng/ml bFGF and 0.5X penicillin-streptomycin.
Renew medium daily to sustain optimal growth conditions.2 Preparation of culture Plates Pre-coat 12-well culture plates with 3% Matrigel for 30-60 minutes before cell seeding to promote attachment and support stem cell maintenance.

3
Cell seeding Seed 1-1.5 ml of hPSCs medium onto each well of the pre-coated culture plates.

CPs and Generation of Cholangiocyte Organoids (COs), D19 -D20 11 Day 19
Remove the medium and wash the cells with PBS.Detach the cells by using TrypLE Select CTS and Detach cells using PBS and cell dissociation buffer, followed by mechanical dissociation as necessary.Seed the cells on another new 3% Matrigel coated dish with WE medium supplemented with 10 ng/mL EGF.
Culture organoids in supplemented WE medium with: • 10 ng/mL EGF • 10 µM Y-27632 Maturation of CO Organoids, D21-D28 13 Day 21-28 Renew the medium with WE medium supplemented with EGF (10 ng/mL) every two days until day 28 Perform bi-daily medium changes using WE medium supplemented with EGF (10 ng/mL) to support organoid maturation until day-28.
Supplementary Table S3: List of RT-qPCR Primer Sequence

Supplementary Figure S5 :
Protocol validation using a human ESC line, H9, and another human iPSC line, IBMS-iPSC-01-02.(A) RT-qPCR of hESC-derived CO under 3D culture shows that the mature cholangiocyte markers, SCTR, CFTR, and CK19, were significantly increased compared to MEF. (B) Immunofluorescence staining of human ESC-derived COs shows a positive for CK19.(C) IF staining shows the morphology of COs derived from IBMS-iPSC-01-02 and iNFB3, which also indicates positive for CFTR and CK19 and has the lumen inside the organoid.(D) Rhodamine-123 fluorescence intensity after Verapamil and DMSO treatment on human PSC-derived COs.Rhodamine-123 fluorescence quantification was performed by using ImageJ (Schneider et al., 2012).MEF, mouse embryonic fibroblast; DMSO, dimethyl sulfoxide.Supplementary Figure S6: Expansion of human iPSC-derived CO under 3D culture.(A) human iPSC-derived COs forming tubular-like structures under 3D culture expansion (P2-3).(B) IF staining for Ki67 shows that in long-term culture after passaging, human iPSC-derived COs decrease their proliferation capacity, which is evaluated by the decrease of Ki67 positive cells.
centrifuge by using 400G to retain the cells.Culture cells on the T75 flask for one day.Collect dissociated cells, and resuspend in Matrigel/WE medium supplemented with: • 10 ng/mL EGF • 10 µM Y-27632 On the next day, take the cells cultured on the T75 dish and centrifuge for 400g in 5 minutes.Cultured the cells in WE medium supplemented with: • 10 ng/mL EGF • 10 µM Y-27632 Seed the cells/Matrigel suspension in the in pre-prepared 12-well plates.Allow the Matrigel dome to solidify for approximately 10 minutes.Next, place the Matrigel dome in an inverted position wihin a 37°C incubator and incubate for around 20 minutes to facilitate cell floating inside the dome.Put the Matrigel dome to the 37oC incubator on inverted position and wait for 15-20 minutes to let the cells floating on the Matrigel dome.Once the Matrigel dome has solidified, add the WE medium supplemented with: • 10 ng/mL EGF • 10 µM Y-27632 12

to Ventral Foregut Endoderm (VFE), D5-D8 8
Replace the differentiation medium with the RPMI/B27 medium supplemented with only high dose of:• 100 ng/mL activin A • 80 ng/mL bFGF to support endodermal lineage maturation and monitor expression of endodermal markers.DE Ensure proper clump size during mechanical dissociation to facilitate subsequent differentiation.4 Pre-differentiation Cultivation Allow cells to acclimate and proliferate for two days prior to commencing the differentiation process.Day 5-8 Cultivate cells in RPMI/B27 medium supplemented with lower 50 ng/mL activin A to steer DE toward a VFE fate, fostering further differentiation and lineage specification.